In biological systems, most processes are under the control of a variety of regulatory mechanisms. For example, the glyoxylate bypass of Escherichia coli is controlled by the phosphorylation of isocitrate dehydrogenase (IDH) and by the regulation of the expression of the glyoxylate bypass operon. Our long-term goal is to characterize these regulatory mechanisms and to determine how they are coordinated. The IDH phosphorylation cycle is catalyzed by a single, bifunctional protein: IDH kinase/phosphatase. Under this proposal, we will study the structure of this bifunctional protein and determine how it specifically recognizes and phosphorylates IDH. In pursuit of these goals, we will characterize the effects of mutations in the genes which encode IDH kinase/phosphatase and IDH. We will also pursue X-ray crystallographic analyses of these proteins and of the complexes which they form. The glyoxylate bypass operon encodes the regulatory and metabolic enzymes of the glyoxylate bypass. Expression of this operon responds to a wide variety of carbon sources and to the availability of molecular oxygen. A goal of this project is to identify the mechanisms by which the expression of this operon is controlled. Under this proposal, we will focus on the repressor protein encoded by iclR. We will also evaluate the possible roles of a variety of other trans-acting factors.